Certain N-substituted 3-oximino-2,4-dioxoquinolin-2,4-(1H)diones useful for treating viral infections

ABSTRACT

Compounds useful as antiviral agents against DNA containing viruses, such as herpes group viruses, are disclosed. The compounds are selected from the group consisting of: ##STR1## and their pharmaceutically acceptable salts and solvates. Pharmaceutical compositions containing compounds represented by Formula 1.00 are disclosed. Also disclosed are methods of treating a viral infection using compounds represented by Formula 1.00 
     The compounds of Formula 1.00 are also useful as intermediates to compounds of: ##STR2## Compounds of Formula 1.0 are useful as antiviral agents against the same viruses that the compounds of Formula 1.00 are useful against.

REFERENCE TO RELATED APPLICATIONS

This application is related to application Ser. No. 07/579,125 filedSep. 7, 1990, now abandoned.

BACKGROUND

This invention relates to compounds having antiviral activity,pharmaceutical compositions thereof, and methods of treatment utilizingthe compositions. In particular, this invention is related to compoundshaving antiviral activity against herpes group viruses, pharmaceuticalcompositions containing the compounds, and methods of treating herpesgroup viruses using the pharmaceutical compositions.

There are four seperate herpes group viruses which infect and causedisease in humans. These are (1) herpes simplex virus 1 and 2 (HSV-1 andHSV-2, respectively); (2) cytomegalovirus (CMV); (3) varicella-zostervirus (VZ); and (4) Epstein-Barr virus (EB). Examples of diseasesassociated with herpes simplex virus infection include herpes labialis,genital herpes(herpes progenitalis), neonatal herpes, herpetickeratitis, eczema herpecticum, disseminated herpes, occupational herpes,herpectic gingivostomatitis, meningitis (aseptic), and encephalitis.

VZ virus is associated with chicken-pox (varicella) and shingles(zoster) in humans.

CMV is wide spread in humans and numerous other mammals. A greatmajority of humans CMV infections are subclinical; that is, the primaryinfection occurs with no signs or symptoms. An exception to this is acongenital infection which occassionally gives rise to cytomegalicinclusion body disease in infants. There is also a mononucleosis-likesyndrome caused by the virus.

A great majority of serious cases due to CMV infection come fromrecurring infections in immuno-compromised individuals, such as intransplant patients and in cancer patients. It has been estimated thatsilent CMV infections have occurred in a majority of humans by the timeadulthood is reached.

Examples of drugs used to treat herpes infections include: (1) IUDR(5'-iodo-2'-deoxyuridine); (2) Ara-C(1-[beta-D-arabinofuranosyl]-cytosine); (3) Ara-A(9-beta-D-arabinofuranosyladenine); and (4) Acyclovir(9-[(2-hydroxyethoxy)methyl]guanine). Also Haines et al. (U.S. Pat. No.4,757,088 issued Jul. 12, 1988) discloses that lidocaine(2-(diethylamino)-N-(2,6-dimethylphenyl)acetamide) is an antiviral agentin cell culture against HSV-1 and HSV-2, and is able to treat herpesvirus infections of mammals. Haines et al. also disclose that lidocaineis particularly effective in the treatment of HSV oral and genitallesions in humans. According to Haines et al., the addition ofpantothenic acid or its alcohol and salt forms, dexpanthenol andpantothenate respectively, to lidocaine or lidocaine hydrochloridesignificantly enhances the antiviral activity of those drugs.

In view of current interest in the art for finding useful antiviralagents, in particular, useful agents against herpes group viruses, anynew compounds exhibiting antiviral activity and/or useful for makingcompounds exhibiting antiviral activity would be a welcome contributionto the art. This invention provides such a contribution.

SUMMARY OF THE INVENTION

This invention provides compounds which are intermeiates to antiviralcompounds and which are themselves useful, as are the antiviralcompounds produced from the intermediates, as antiviral agents againstDNA containing viruses such as herpes group viruses. In particular, thecompounds of this invention are useful against HSV-1 and HSV-2 and mayalso prove useful against CMV and EB.

The compounds of this invention are advantageous over known compoundsbecause they inhibit early events in the viral replication.

One embodiment of this invention provides compounds of: ##STR3##wherein: (A) X is selected from the group consisting of N, O, S, and C;

(B) m is an integer from 0 to 4;

(C) Each R₂ for each m is independently selected from the groupconsisting of:

(1) alkyl;

(2) alkoxy;

(3) aryloxy;

(4) aryl;

(5) aralkyloxy;

(6) halogen atoms selected from the group consisting of: F, Cl, Br andI;

(7) ##STR4## wherein R₅ is selected from the group consisting of: alkyl,alkaryl, alkenyl, heteroalkyl, and heteroaryl;

(8)

    --N(R.sub.6).sub.2

wherein each R₆ is independently selected from the group consisting of:H, alkyl, aryl and R₅ C(O)-- wherein R₅ is as above defined;

(9)

    --OH;

(10)

    --CH.sub.2 OH;

(11) ##STR5## (12) ##STR6## wherein R₇ is selected from the groupconsisting of: alkyl and aryl; (13)

    --SO.sub.3 H;

(14)

    --SO.sub.2 NHR.sub.8

wherein R₈ is selected from the group consisting of: alkyl, aryl and H;

(15)

    --PO.sub.3 H;

and

(16)

    --PO(OR.sub.9).sub.2

wherein R₉ is selected from the group consisting of: alkyl and aryl;

(17) --OPO₃ H;

(18) --OP(OR₉)₂ wherein R₉ is as above defined; and

(19) --CF₃ ;

(D) n is:

(1) 1 when X is N;

(2) 0 when X is S;

(3) 0 when X is O; and

(4) 2 when X is C, and each R₃ is the same or different;

(E) R₃ is selected from the group consisting of:

(1) alkyl;

(2) aralkyl:

(3) aryl;

(4) substituted aryl;

(5) alkaryl;

(6) alkyl heteroaryl;

(7) alkyloxyalkyloxyaryl;

(8) --(CH₂)_(a) R₁₀ wherein a is an integer of 1 to 6 and R₁₀ isselected from the group consisting of:

(a) --C(O)OR₁₁ is selected from the group consisting of alkyl, alkenyl,and H;

(b) --N(R₁₁)₂ wherein each R₁₁ is the same or different, and R₁₁ is asdefined above;

(c) --R₁₁ wherein R₁₁ is as defined above;

(d) --OR₁₁ wherein R₁₁ is as defined above;

(9) H; and

(10) --OR₁₂ wherein R₁₂ is selected from the group consisting of H,alkyl, alkaryl, alkenyl, heteroaryl, and heteroalky.

Those skilled in the art with appreciate that compounds of Formula 1.00can also exist in the tautomeric form represented by Formula 1.0:##STR7## and that reference herein to Formula 1.00 includes reference toFormula 1.01.

Another embodiment of this invention provides pharmaceuticalcompositions comprising an effective amount of a pharmaceuticallyacceptable carrier and an effective amount of a compound of thisinvention. Preferably the compound is selected from the group ofcompounds represented by Formulas 1.02 to 1.07 defined below. Thepharmaceutical compositions are useful in treating viral infections in apatient in need of such treatment.

In yet another embodiment this invention provides a method of treating apatient having a viral infection by administering to such a patient aneffective amount of a compound of this invention. Generally, thecompound is administered as one of the pharmaceutical compositions ofthis invention. Examples of viral infections treatable in accordancewith the methods of this invention include the DNA containing virusessuch as the herpes viruses discussed above (e.g., HSV-1, HSV-2, CMV, VZ,EB, and the like).

The compounds of Formula 1.00 are useful as intermediates to thecompounds of Formula 1.0: ##STR8## wherein: (A) X, m, R₂, R₃ and n areas defined above; and

(B) R₁ is selected from the group consisting of:

(1) alkyl;

(2) aryl;

(3) acyl of the formula --C(O)R₄ wherein R₄ is selected from the groupconsisting of: H, aryl, alkaryl, alkenyl, --NH₂, --NHR₅, --N(R₅)₂,heteroalkyl, heteroaryl, and substituted alkyl wherein R₅ is selectedfrom the group consisting of: alkyl, alkaryl, alkenyl, heteroalkyl, andheteroaryl;

(4) heteroaryl; and

(5) H.

Compounds of Formula 1.0 are useful in treating the same viralinfections disclosed as being treatable by the compounds of Formula1.00.

DETAILED DESCRIPTION OF THE INVENTION

When used herein, the terms listed below have the scope indicated,unless indicated otherwise.

Alkaryl--represents an aryl group, as defined below, in which an alkylgroup, as defined below, is substituted for one of the aryl H atoms. Thearyl group may contain additional substituents selected from the groupconsisting of: halogen atoms (e.g, Cl, Br, F, and/or I), alkoxy, alkyl,and amino. Representative examples include: CH₃ phenyl-, CH₃ CH₂ phenyl-and the like.

Alkenyl (alkylene)--represents straight and branched carbon chainshaving at least one carbon to carbon double bond and preferably havingfrom 2 to 6 carbon atoms. Preferably the alkenyl substituent has from 1to 2 double bonds. Representative examples include vinyl, allyl, butenyland the like.

Alkoxy--represents an alkyl radical attached to a molecule through anoxygen atom (--O-alkyl). Representative examples include methoxy, ethoxyand the like.

Alkynyl--represents a straight or branched hydrocarbon chain having atleast one carbon-to-carbon triple bond, and having from 3 to 8 carbonatoms with from 3 to 6 carbon atoms being preferred. Representativeexamples include propynyl, butynyl and the like.

Alkyl--represents straight or branched carbon chains, which contain from1 to 6 carbon atoms. Representative examples include methyl, ethyl,propyl and the like.

Alkyl heteroaryl(alkheteroaryl)--represents a heteroaryl group, asdefined below, wherein an alkyl group, as defined above, is substitutedfor one of the aryl H atoms. Representative examples includepyridylmethyl, furylmethyl and the like.

Alkyloxyalkyloxyaryl--represents a group wherein an alkyl group isjoined through an oxygen atom to another alkyl group which in turn isjoined through an oxygen atom to an aryl group wherein the point ofattachment to the aryl group is at a ring carbon. Alkyl is as definedabove and aryl is as defined below. The aryl group may containadditional substituents selected from the group consisting of: halogenatoms (e.g., Cl, Br, F, and/or I), alkoxy, alkyl, and amino.Representative examples include phenoxypropyloxymethyl,phenoxyethoxymethyl and the like.

Aralkyl--represents an alkyl group as defined above in which an arylgroup as defined below is substituted for one of the alkyl hydrogenatoms. Representative examples include --CH₂ phenyl, --CH₂ CH₂ phenyl,4-hydroxybenzyl, 4-t-butyldimethylsilyloxybenzyl, and the like.

Aralkyloxy--represents an aralkyl group as defined above, which isattached to a molecule by an oxygen atom (aralkyl-O--). The aryl groupmay contain additional substitutents selected from the group consistingof: halogen atoms (e.g., Cl, Br, F, and/or I), alkoxy, alkyl, and amino.Representative examples include benzyloxy, phenethoxy, and the like.

Aryl--represents a mono or bi-cyclic aromatic system. Examples ofpreferred aryl groups include those having from 6 to 14 carbon atoms.Representative examples include phenyl, 1-naphthyl, 2-naphthyl andindanyl. The aryl group may contain additional substituents selectedfrom the group consisting of: halogen atoms (e.g., Cl, Br, F, and/or I),alkoxy, alkyl, and amino.

Aryloxy--represents an aryl group as defined above, which is attachedthrough an oxygen atom (aryl-O--). The aryl may contain additionalsubstituents selected from the group consisting of: halogen atoms (e.g.,Cl, Br, F, and/or I), alkoxy, alkyl, and amino. Representative examplesinclude phenoxy, naphthyloxy, and the like.

Heteroalkyl--represents an alkyl group, as defined above, wherein one ormore heteroatoms are substituted for one or more of the alkyl H atoms.The heteroatoms are independently selected from the group consisting of:O, S, and N. Representative examples of heteroalkyl groups includehydroxyethyl, aminoethyl, mercaptoethyl, and the like.

Heteroaryl (including the heteroaryl portion ofheteroarylmethyl)--represents aromatic systems having at least one O, Sand/or N heteroatom in the ring structure. Examples of preferredheteroaryl groups include those containing from 3 to 14 carbon atoms.Representative examples of heteroaryl groups include but are not limitedto: 2-, 3- or 4-pyridyl, 2- or 3-furyl, 2- or 3-thienyl, 2-, 4- or5-thiazolyl, 2-, 4- or 5-imidazolyl, 2-, 4- or 5-pyrimidinyl,2-pyrazinyl, 3- or 4-pyridazinyl, 3-, 5- or 6-[1,2,4-triazinyl], 3- or5-[1,2,4-thiadiazolyl], 2-, 3-, 4-, 5-, 6- or 7-benzofuranyl, 2-, 3-,4-, 5-, 6- or 7-indolyl, 3-, 4- or 5-pyrazolyl, 2-, 4- or 5-oxazolyl,2-or 3-pyrrolyl, 2- or 3-N-methylpyrrolyl, and the like.

Substituted alkyl--represents an alkyl group, as defined above, whereinone or more of the alkyl H atoms are replaced with groups selected fromthe group consisting of: alkyl, aryl, heteroaryl, --OH, --O-alkyl,--NH₂, --N(alkyl)₂ wherein each alkyl group is the same or different,--S-alkyl, --C(O)O-alkyl, --C(O)H, --NHC(NH)NH₂ (wherein the C(NH)portion represents C═NH), --C(O)NH₂, --OC(O)NH₂, NO₂ and --NHC(O)-alkyl,wherein alkyl, aryl, and heteroaryl are as above defined.

Substituted aryl--represents an aryl group, as defined above, whereinone of more of the H atoms attached to the ring carbon atoms arereplaced by groups independently selected from the group consisting of:halo, alkyl, hydroxy, alkoxy, phenoxy, amino, alkylamino, anddialkylamino. Preferred substituted aryl groups are substituted phenylgroups.

Also, as used herein, unless indicated otherwise, C(O) represents C═O

R₁ is preferably selected from the group consisting of: alkyl, acyl, andH. When R₁ is acyl, i.e., --C(O)R₄, R₄ is preferably selected from thegroup consisting of: alkyl, aryl, and heteroaryl (including substitutedheteroaryl). Most preferably R₄ is selected from the group consistingof: methyl, ethyl, phenyl, 2-pyrrolyl, N-methyl-2-pyrrolyl,5-bromo-2-furanyl, 4-pyridyl(4-pyridinyl), and 2-thiophenyl (2-thienyl).

Representative examples of R₁ include, but are not limited to:

(1) H;

(2) methyl;

(3) pyrrol-2-ylcarbonyl (pyrrol-2-carbonyl) ##STR9## (4)N-methylpyrrol-2-ylcarbonyl(N-methylpyrrol-2-carbonyl) ##STR10## (5)2-thiophenecarbonyl (2-thienylcarbonyl) ##STR11## (6)5-bromo-furanylcarbonyl ##STR12## (7) isonicotinoyl ##STR13## (8)benzoyl ##STR14## (9) acetyl ##STR15## (10) propionyl ##STR16## (11) andthe like.

Examples of R₂ include, but are not limited to:

(1)

    --CH.sub.3 ;

(2)

    --OCH.sub.3 ;

(3) ##STR17## (4) ##STR18## (5) Cl; (6) F and;

(7) I; and

(8) --CF₃.

Examples of R₃ include, but are not limited to:

(1)

    --CH.sub.3 ;

(2)

    --C.sub.6 H.sub.13 ;

(3)

    --C.sub.7 H.sub.5

(4) ##STR19## (5) ##STR20## (6) ##STR21## (7) ##STR22## (8) ##STR23##and (9) ##STR24##

Preferably R₃ is selected from the group consisting of: methyl, heptyl,phenyl and benzyl.

Preferably X is nitrogen.

Examples of compounds of Formula 1.00 include, but are not limited to,compounds of Formulas: ##STR25##

Compounds of this invention are used to make compounds of Formula 1.0.Examples of compounds of Formula 1.0 include, but are not limited to:##STR26##

Preferred compounds of this invention are selected from the groupconsisting of: Formulas 1.02, 1.03, and 1.04.

Certain compounds of this invention may exist in isomeric forms. Theinvention contemplates all such isomers both in pure form and inadmixture, including racemic mixtures.

Certain compounds of the invention can exist in unsolvated as well assolvated forms, including hydrated forms, e.g., hemihydrate. In generalsuch as water, ethanol and the like are equivalent to the unsolvatedforms for purposes of the invention.

Certain compounds of the invention will be acidic in nature, e.g., thosecompounds which possess a carboxyl or phenolic hydroxyl group. Thesecompounds may form pharmaceutically acceptable salts. Examples of suchsalts are the sodium, potassium, calcium, and aluminum. salts. Alsocontemplated are salts formed with pharmaceutically acceptable aminessuch as ammonia, alkylamines, hydroxyalkylamines, N-methylglucamine andthe like.

Certain compounds of the invention, e.g., those with a basic aminegroup, also form pharmaceutically acceptable salts with organic andinorganic acids. Examples of suitable acids for salt formation arehydrochloric, sulfuric, phosphoric, acetic, citric, oxalic, malonic,salicylic, malic, fumaric, succinic, ascorbic, maleic, methanesulfonicand other mineral and carboxylic acids well known to those skilled inthe are. The salts are prepared by contacting the free base form with asufficient amount of the desired acid to produce a salt in theconventional manner. The free base forms may be regenerated by treatingthe salt with a suitable dilute aqueous base solution such as diluteaqueous sodium hydroxide, potassium carbonate, ammonia and sodiumbicarbonate. The free base forms differ from their respective salt formssomewhat in certain physical properties, such as solubility in polarsolvents, but the salts are otherwise equivalent to their respectivefree base forms for purposes of this invention.

The compounds of Formula 1.0 can be prepared by the processes describedbelow. In these processes the substituents are as described above,unless indicated otherwise. Those skilled in the art will appreciatethat the processes described below the reactions are carried out at atemperature high enough to allow the reaction to proceed at a reasonablerate, but not so high as to cause undue degradation of reactants and/orproducts. Those skilled in the art will also appreciate that in thefollowing reactions the desired products may be isolated by techniqueswell known in the art such as distillation, column chromatography,recrystallization, and the like.

The compounds of Formula 1.00 can be prepared in accordance with thereactions set forth in Scheme I. The abbreviation "EQ" as usedhereinafter stands for Equation. ##STR27##

EQ 1.0, 1.1, and 1.2 represent reactions well known to those skilled inthe art, see, for example: G. M. Coppola, et al., Synthesis, 505 (1980);the disclosure of which is incorporated herein by reference thereto.

In EQ 1.0 a suitable 2-aminobenzoic acid (2.0) in a water and 2N Hclsolution is reacted with trichloromethyl chloroformate to form anisatoic anhydride (2.1). The aminobenzoic acid (2.0) used will have theappropriate R₂ substitutent group to give the desired ultimate endproduct. R₂ and m are as defined above.

In EQ 1.1, the isatoic anhydride (2.1) is reacted with a suitable halideof R₃ (wherein R₃ and n are as above defined) to produce the desiredsubstituted isatoic anhydride (2.2).

In EQ 1.2, the isatoic anhydride is reacted with the anion derived froma malonate ester to produce the quinolone(2.3). R₁₃ is ethyl when ehtylmalonate is used.

In EQ 1.3, the quinolinone (2.3) is decarboxylated to produce thequinolinone (2.4). Generally this reaction is carried out by procedureswell known in the art--see, for example, G. M. Coppola et al., J. Org.Chem., 41, 825 (1976), the disclosure of which is incorporated herein byreference thereto.

In EQ 1.4, the compound of Formula (2.4) is converted to the compound ofFormula (1.00) by following standard reaction conditions fornitrosation, see, for example, J. March, Adv. Org. Chem., John Wiley &Sons Publishers, 1985, p. 535, the disclosure of which is incorporatedherein by reference thereto.

The compounds of Formula 1.00 can then be converted to compounds ofFormula 1.0 in accordance with Equations 1.5 and 1.6. ##STR28##

In EQ 1.5, the compound of Formula 1.00 is reductively hydrolyzed toproduce a compound of Formula 2.5. The reaction is carried out bystirring a mixture of a compound of Formula 1.00 and a hydrogenationcatalyst in an acidic solvent mixture under hydrogen.

The hydrogenation catalyst used in EQ 1.5 may be of the palladium,platinum or nickel type; preferably the catalyst is 5-20% palladium oncarboxy; most preferably the catalyst is 10% palladium on carbon. Theamount of catalyst used is from about 0.01 to about 0.5 parts by weightbased on the weight of the compound (substrate) of Formula 1.00;preferably about 0.1 to about 0.5 parts by weight and most preferablyabout 0.3 parts by weight of catalyst is used.

The acidic solvent used in the reaction of EQ 1.5 comprises a mixture ofan organic solvent, such as an alcohol (e.g., ethanol, methanol,propanol, isopropanol, and the like), glacial acetic acid,tetrahydrofuran, dimethylformamide and the like, containing a mineralacid such as hydrochloric or sulfuric acid. Preferably the solvent isethanol or glacial acetic acid containing aqueous hydrochloric orsulfuric acid; most preferably the solvent comprises a mixture of 100parts of ethanol and 50 parts of 2N-hydrochloric acid for 1 part ofsubstrate (compound) of Formula 1.00 (Vol/Wt).

The hydrogenation in EQ 1.5 is carried out at pressures ranging fromatmospheric pressure to several p.s.i.; preferably the reaction iscarried out at atmospheric pressure to 15 p.s.i. and most preferably atatmospheric pressure. The reaction is complete when no further uptake ofhydrogen is observed.

In EQ 1.6, the compound of Formula (2.5) is reacted with an appropriatehalide of R₁ in order to produce the desired ether, or with anappropriate acyl halide to produce an ester at C-3 and C-4. Thereactions are carried out by procedures well known in the art; forexample, see J. March, Adv. Org. Chem., cited above, Section 0.14, p.342, for producing the ethers, and see J. March, Adv. Org. Chem., citedabove, Section 0.22, p. 346, for producing the esters, the disclosuresof each being incorporated herein by reference thereto.

The compounds of this invention can be administered in any number ofconventional dosage forms, e.g., topical, oral, parenteral, rectal,transdermal, inhalation and the like. Oral or rectal dosage formsinclude capsules, tablets, pills, poweders, cachets and suppositories.Liquid oral dosage forms include solutions and suspensions. Parenteralpreparations include sterile solutions and suspensions. Inhalationadministration can be in the form of a nasal or oral spray, or byinsufflation. Topical dosage forms can be creams, ointments, lotions,transdermal devices (e.g., of the conventional patch or matrix type) andthe like.

The formulations and pharmaceutical compositions contemplated by theabove dosage forms can be prepared with conventional pharmaceuticallyacceptable excipients and additives, using conventional techniques. Suchpharmaceutically acceptable excipients and additives are intended toinclude carriers, binders, flavorings, buffers, thickeners, coloringagents, stabilizing agents, emulsifying agents, dispersing agents,suspending agents, perfumes, preservatives lubricants, etc.

Suitable pharmaceutical acceptable solid carriers are magnesiumcarbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin,starch, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose, low melting waxes, cocoa butter and the like.Capsules can be made wherein the active compound is inserted intopharmaceutically acceptable capsules as a carrier. The active compoundsof this invention can be mixed with pharmaceutically acceptableexcipients or be used in finely divided powder form without excipientsfor inclusion into the capsules. Similarly, cachets are included.

Liquid form preparations include solutions, suspensions and emulsionssuch as water or water-propylene glycol solutions for parenteralinjection. Liquid preparations can also be formulated in solution inpolyethylene glycol and/or propylene glycol, which may contain water.Aqueous solutions suitable for oral use can be prepared by adding theactive component in water and adding suitable colorants, flavors,stabilizing, sweetening, solubilizing and thickening agents as desired.Aqueous suspensions suitable for oral use can be made by dispersing theactive component in finely divided form in water with viscous material,i.e., pharmaceutically acceptable natural or synthetic gums, resins,methylcellulose, sodium carboxymethylcellulose and other well-knownsuspending agents.

Formulations for topical application may include the above liquid forms,as well as creams, aerosols, sprays, dusts, powders, lotions andointments which are prepared by combining an active ingredient accordingto this invention with conventional pharmaceutical acceptable diluentsand carriers commonly used in topical dry, liquid, cream and aerosolformulations. Ointment and creams may, for example, be formulated withan aqueous or oily base with the addition of suitable thickening and/orgelling agents. Such bases may, thus, for example, include water and/oran oil such as liquid paraffin or a vegetable oil such as peanut oil orcastor oil. Thickening agents which may be used according to the natureof the base include soft paraffin, aluminum stearate, cetostearylalcohol, propylene glycol, polyethylene glycols, woolfat, hydrogenatedlanolin, beeswax, etc.

Lotions may be formulations with an aqueous or oil base and will, ingeneral, also include one or more of pharmaceutically acceptablestabilizing agents, emulsifying agents, dispersing agents, suspendingagents, thickening agents, coloring agents, perfumes and the like.

Powders may be formed with the aid of any suitable pharmaceuticallyacceptable powder base, e.g., talc, lactose, starch, etc. Drops may beformulated with an aqueous base or non-aqueous base also comprising oneor more pharmaceutically acceptable dispersing agents, suspendingagents, solubilizing agents, etc.

The topical pharmaceutical compositions may also include one or morepreservatives or bacteriostatic agents, e.g., methyl hydroxybenzoate,propyl hydroxybenzoate, chlorocresol, benzalkonium chlorides, etc.

The topical pharmaceutical compositions may also contain an activecompound of this invention in combination with other active ingredientssuch as antimicrobial agents, particularly antibiotics, anesthetics,analgesics and antipruritic agents.

Also included are solid form preparations which are intended to beconverted, shortly before use, to liquid form preparations for eitheroral or parenteral administration. Such liquid forms include solutions,suspensions and emulsions. These particular solid form preparations aremost conventiently provided in unit dose form and as such are used toprovide a single liquid dosage unit. Alternatively, sufficient solid maybe provided so that after conversion to liquid form, multiple individualliquid doses may be obtained by measuring predetermined volumes of theliquid form preparation as with a syringe, teaspoon or other volumetriccontainer. When multiple liquid doses are so prepared, it is preferredto maintain the unused portion of said liquid doses under conditionswhich retard possible decomposition. The solid form preparationsintended to be converted to liquid form may contain, in addition to theactive material, pharmaceutically acceptable flavorants, colorants,stabilizers, buffers, artificial and natural sweeteners, dispersants,thickeners, solubilizing agents and the like. The solvent utilized forpreparing the liquid form preparation may be water, isotonic water,ethanol, glycerine, propylene glycol and the like as well as mixturesthereof. Naturally, the solvent utilized will be chosen with regard tothe route of administration, for example, liquid preparations containinglarge amounts of ethanol are not suitable for parenteral use.

The compounds of this invention may also be deliverable transdermallyfor systemic distribution. The transdermal compositions can take theform of creams, lotions and/or emulsions and can be included in atransdermal patch of the matrix or reservoir type as are conventional inthe art for this purpose.

The compounds of this invention may be administered by any conventionalmode of administration, by employing an antiviral effective amount of acompound of this invention for such mode. The dosages may be varieddepending upon the requirements of the patient in the judgment of theattending clinician, the severity of the condition being treated and theparticular compound being employed. Determination of the proper dosagefor a particular situation is within the skill of the art. Treatment canbe initiated with smaller dosages which are less than the optimum doseof the compound. Thereafter, the dosage should be increased by smallincrements until the optimum effect under the circumstances is reached.For convenience, the total daily dosage may be divided and administeredin portions during the day if desired.

Thus, depending on the mode, dosages of from about 0.1 to about 100mg/kg of body weight per day may be administered to provide antiviralactivity. For example, when administered orally doses of from about 20to about 60 mg/kg of body weight may be used; and when administeredparenterally, e.g., intravenously, dosages of from about 5 to about 20mg/kg body weight may be used.

When administered topically, the amount of compound administered varieswidely with the amount of skin being treated, as well as with theconcentration of active ingredient applied to the affected area.Preferrably, topical compositions contain from about 0.10 to about 10percent by weight of the active ingredient and are applied as neededaccording to the judgment of the attending clinician. When administeredrectally, the compounds of this invention may be administered in dailydoses ranging from about 0.1 mg/kg to about 100 mg/kg.

The dosage to be administered and the route of administration dependsupon the particular compound used, the age and general health of thepatient and the severity of the viral condition. Thus, the doseultimately decided upon must be left to the judgment of a trainedhealth-care practitioner.

EXAMPLES

The following examples are illustrative only and should not be construedas limiting the invention in any way. Those skilled in the art willappreciate that variations are possible which are within the spirit andscope of the appended claims.

In the examples, Ar represents aromatic.

PREPARATION A 1-Benzyl-4-Hydroxy-2(1H)-Quinolinone

(1) A solution of isatoic anhydride (4.1 gm) in DMF (30 ml) was addeddropwise to a stirred suspension of 60% sodium hydride (1.0 gm) in DMF(20 ml) under nitrogen atmosphere. The reaction mixture was then warmedto 45° C. and stirred until hydrogen evolution ceased. The reactionmixture was then cooled and a solution of benzyl bromide (4.4 gm) in DMF(10 ml) was added slowly. Stirring was continued for one hour at roomtemperature and the solution was then evaporated under reduced pressureat 45° C. The resulting solid was removed by filtration and the filtratewas evaporated to give 1-benzylisatoic anhydride.

(2) Diethyl malonate (4.07 gm) in dimethyl acetamide (DMA) (10 ml) wasadded dropwise to a stirred suspension of 60% sodium hydride (1.01 gm)in the same solvent (10 ml), under a nitrogen atmosphere, in an oil bathat 25° C. After hydrogen evolution ceased, the temperature was raised to80° C. while adding a solution of 1-benzylisatoic anhydride (4.2 gm) inDMA (50 ml). After carbon dioxide evolution ceased, the reaction mixturewas heated at 120° C. for 17 hours and then concentrated under reducedpressure to a volume of 25 ml, and then diluted with water (50 ml). Themilky solution was washed with ether, the aqueous layer was acidifiedwith mineral acid to pH3 and the resulting crystalline product1-benzyl-3-carbethoxy-2(1H)-quinolinone was added to 2N sodium hydroxide(150 ml). The mixture was refluxed for 4 hrs and the resulting solutionwas then cooled, acidified with mineral acid to pH3 and the solid wasfiltered, dried and crystallized from ethyl acetate/hexane to give thetitle compound (4.0 gm). That the expected product was obtained wasconfirmed by the spectral data: MS: m/e 251 (M.⁺); NMR (DMSO): δ5.47(s,2H,CH₂ Ar), 6.03(s,1H,═CH), 11.6 (s,1H,OH) ppm.

PREPARATION B 1-Heptyl-4-Hydroxy-2(1H)-Quinolinone

Obtained by using heptyl bromide in Step (1) of Preparation A. That theexpected product was obtained was confirmed by the spectral data: MS:m/e 259 (M.⁺); NMR (DMSO): δ0.9(t, 3H, CH₃ --CH₂), 4.06 (t, 2H, N--CH₂),6.18 (s, 1H, ═CH), 11.87 (br, 1H, OH) ppm.

PREPARATION C 1-Phenyl-4-Hydroxy-1 (1H)-Quinoline

Obtained by starting with N-phenyl-isatoic anhydride and using theprocedure described in Step (2) of Preparation A. That the expectedproduct was obtained was confirmed by the spectral data: MS: m/e 237(M.⁺); NMR (DMSO): δ5.92 (s, 1H, ═CH), 11.60 (s, 1H, OH) ppm.

EXAMPLE 1 1-Methyl-3-Oximino-Quinolin-2,4(1H)-Dione (Formula 1.02)

Glacial acetic acid (2.7 ml) was added with stirring to a solution of1-methyl-4-hydroxy-2(1H)-quinolinone (1.75 gm, see G. M. Copploa, J.Org. Chem., 41, 825, (1976), already cited above) and sodium nitrite(0.8 gm) in a solvent mixture consisting of methanol (17.5 ml), ethylacetate (20 ml) and water (10 ml) under nitrogen atmosphere. Thereaction mixture was stirred for 1.5 hrs at room temperature and theorganic solvents were then removed under reduced pressure. The resultingsuspension was filtered and the solid washed with water to give thetitle compound. That the expected product was obtained was confirmed bythe spectral data: MS: m/e 204 (M.⁺); NMR (DMSO): δ3.44 (s, 3H, CH₃--N), 15.34 (br, 1H, ═NOH) ppm.

EXAMPLE 2 1-Benzyl-3-Oximino-Quinoline-2,4(1H)-Dione (Formula 1.03)

Obtained by nitrosation of 1-benzyl-4-hydroxy-2(1H)-quinolinone(Preparation A) using the procedure described in Example 1. That theexpected product was obtained was confirmed by the spectral data: MS:m/e 280 (M.⁺); NMR (DMSO): δ5.35(s, 2H, CH₂ --Ar), 15.3 (br, 1H, ═NOH)ppm.

EXAMPLE 3 1-Heptyl-3-Oximino-Quinolin-2,4(1H)-Dione (Formula 1.04)

Obtained by nitrosation of 1-heptyl-4-hydroxy-2(1H)-quinolinone(Preparation B) using the procedure described in Example 1. That theexpected product was obtained was confirmed by the spectral data: MS:m/e 288 (M.⁺); NMR (DMSO): δ0.85 (t, 3H, CH₃ --), 1.28 (br, 6H, --CH₂--), 1.62 (br, 2H, --CH₂ --), 4.02(t, 2H, CH₃ --N), 15.37(br,1H,═NOH)ppm.

EXAMPLE 4 1-Phenyl-3-Oximino-Quinolin-2,4(1H)-Dione (Formula 1.05)

Obtained by nitrosation of 1-phenyl-4-hydroxy-2(1H)-quinolinone(Preparation C) using the procedure described in Example 1. That theexpected product was obtained was confirmed by the spectral data: MS:m/e 266 (M.⁺); NMR (DMSO): δ15.19 (br, 1H, ═NOH) ppm.

EXAMPLE 5 3-Oximino-7-Hydroxy-8-Methyl-Benzopyran-2,4-Dione (Formula1.06)

Obtained by nitrosation of 4,7-dihydroxy-8-methylcoumarin (JACS, 80,140, 1958, the disclosure of which is incorporated herein by referencethereto) using the procedure described in Example 1. That the expectedproduct was obtained was comfirmed by the spectral data: MS: m/e 221(M.⁺).

EXAMPLE 6 3-Oximino-4-Oxo-Dihydro-1,2-Benzothiopyrone (Formula 1.07)

If the following procedure were to be followed then the title compoundwould be obtained.

Step (1): Preparation of 4-Hydroxy-1,2-Benzothiopyrone.

A solution of thiosalicylic acid (3.0 g) in methanol (30 ml) is cooledin an ice bath and dry hydrogen chloride is bubbled in for 15 minutes.The solution is allowed to stand at room temperature for 24 hours andthen evaporated. The resulting methyl thiosalicylate is dissolved inpyridine (20 ml) containing acetic anhydride (5 ml). After 24 hrs thereaction mixture is diluted with ethyl acetate, washed several timeswith water, dried and evaporated. The resulting acetylthiosalicylic acidmethyl ester is dissolved in dimethylformamide (20 ml), cooled to 0° C.and 60% sodium hydride (0.8 g) is added while stirring. The reactionmixture is then stirred room temperature for 12 hours, concentrated tohalf volume under reduced pressure, diluted with water and acidified topH3 with mineral acid and filtered to give the title compound.

Step (2): Preparation of 3-Oximino-4-oxo-dihydro-1,2-benzothiopyrone.

Is obtained by nitrosation of 4-hydroxy-1,2-benzothipyrone (Step 1)using the procedure described in Example 1.

INTERMEDIATE TO FINAL PRODUCT

The compounds of the invention (Formula 1.00), as stated above, areintermediates to compounds of Formula 1.0 (defined above). The followingexamples illustrate the use of the compounds of Formula 1.00 to producethe compounds of Formula 1.0.

EXAMPLE 7 1-Methyl-3,4-Dihydroxy-2(1H)-Quinolinone; (Formula 1.1 fromFormula 1.02)

A suspension of 1-methyl-3-oximino-quinolin-2,4(1H)-dione (1.7 gm,Formula 1.02, Example 1) and 10% palladium on charcoal (0.5 gm) in amixture of ethanol (90 ml) and 2N hydrochloric acid (40 ml) washydrogenated at atmospheric conditions. After 2.5 hrs no further uptakeof hydrogen (510 ml) was observed. The reaction mixture was filtered andthe solid cake was extracted with hot glacial acetic acid (2×100 ml).The combined filtrates were evaporated under reduced pressure and theproduce was crystallized from acetic acid/water to give the titlecompound (1.6 gm). That the expected product was obtained was confirmedby the spectral data: MS (FAB): m/e 192 (M.⁺ +1); NMR (DMSO): δ3.66 (s,3H, CH₃ --N), 8.79 (s, 1H, OH), 10.16 (s, 1H, OH) ppm.

EXAMPLE 8 1-Benzyl-3,4-Dihydroxy-2(1H)-Quinolinone (Formula 1.2 fromFormula 1.03)

Obtained from 1-benzyl-3-oximino-quinolin-2,4(1H)-dione (Formula 1.03,Example 2) using the hydrolytic reduction procedure described in Example7. That the expected product was obtained was confirmed by the spectraldata: MS: m/e 267 (M.⁺); NMR (DMSO): δ5.59 (s, 2H, CH₂ --Ar), 8.92 (br,1H, OH), 10.32 (br, 1H, OH) ppm.

EXAMPLE 9 1-Heptyl-3,4-Dihydroxy-2(1H)-Quinolinone (Formula 1.3 fromFormula 1.04)

Obtained from 1-heptyl-3-oximino-quinolin-2,4(1H)-dione (Formula 1.04,Example 3) using the hydrolytic reduction procedure described in Example7. That the expected product was obtained was confirmed by the spectraldata: MS: m/e 275 (M.⁺); NMR (DMSO): δ0.84 (t, 3H, CH₃ --), 1.28 (m,8H,--CH₂ --), 1.62 (m, 2H, --CH₂ --), 4.27 (t, 2H, CH₂ --N), 8.76 (d,1H, OH), 10.16 (d, 1H, OH) ppm.

EXAMPLE 10 1-Phenyl-3,4-Dihydroxy-2(1H)-Quinolinone (Formula 1.6 fromFormula 1.05)

Obtained from 1-phenyl-3-oximino-quinolin-2,4(1H)-dione (Formula 1.05,Example 4) using the hydrolytic reduction procedure described in Example7. That the expected product was obtained was confirmed by the spectraldata: MS: m/e 254 (M.⁺ +1).

EXAMPLE 111-Methyl-3,4-di(N-Methyl-Pyrrol-2-carbonyloxy)-2(1H)-Quinolinone(Formula 1.4 from Formula 1.02)

Thionyl chloride (10 ml) was added to N-methyl-pyrrole-2-carboxylic acid(2.5 gm) in ether (10 ml), the mixture was refluxed for 20 mins andevaporated under reduced pressure. The residual oil was azeotroped withbenzene. The resulting acid chloride was dissolved in methylene chloride(10 ml) and added to a solution of1-methyl-3,4-dihydroxy-2(1H)-quinolinone (1.7 gm, Example 7, obtainedfrom Formula 1.02, see Example 1) in pyridine (20 ml). The reactionmixture was stirred overnite at room temperature, then diluted withmethylene chloride, then washed with water, then dried and thenevaporated under reduced pressure. The crude product was then purifiedby chromatography on silica gel using chloroform as the eluting solvent.Fractions containing the major product (silica gel tlc plates; 12.5%ethyl acetate/chloroform solvent system) were combined and crystallizedfrom ethyl acetate to give the title compound (2.24 gm). That theexpected product was obtained was confirmed by the spectral data: MS(FAB): m/e 406 (M.⁺ +1); NMR(CDCl₃): δ3.81 (s, 3H, CH₃ --N), 3.90 (s,3H, CH₃ --N), 3.92 (s, 3H, CH₃ --N), 6.10, 6.19, 6.81, 6.89, 7.12,7.26(m, 6H, Pyrrole) ppm.

EXAMPLE 12 1-Methyl-3,4-di-(Pyrrole-2-carbonyloxy)-2(1H)-Quinolinone(Formula 1.5 from Formula 1.02)

Obtained by acylation of 1-methyl-3,4-dihydroxy-2(1H)-quinolinone(Example 7, obtained from Formula 1.02, see Example 1) withpyrrole-2-carboxylic acid using the procedure described in Example 11.That the expected product was obtained was confirmed by the spectraldata: MS: m/e 377 (M.⁺); NMR(CDCl₃): δ3.79 (s, 3H, CH₃ --N), 6.22 (m,1H, ═CH), 6.34 (m, 1H, ═CH), 6.95 (m, 1H, ═CH), 7.06 (m, 2H, ═CH), 7.21(m, 1H, ═CH), 7.30 (m, 1H, ═CH), 7.44 (d, 1H, ═CH), 7.60 (m, 1H, ═CH),7.72 (d, 1H, ═CH), 9.31 (br, 1H, NH), 9.49 (br, 1H, NH) ppm.

EXAMPLE 13 1-Methyl-3,4-di-(4-bromo-2-furoyloxy)-2(1H)-Quinolinone(Formula 1.7 from Formula 1.02)

Obtained by acylation of 1-methyl-3,4-dihydroxy-2(1H)-quinolinone(Example 7, obtained from Formula 1.02, see Example 1) with 2-bromofuroic acid using the procedure described in Example 11. That theexpected product was obtained was confirmed by the spectral data:MS(FAB): m/e 538 (M.⁺ +1); NMR(CDCl₃): δ3.81 (s, 3H, CH₃ --N), 6.50,6.58, 7.32, 7.44 (d, 4H, Furan) ppm.

EXAMPLE 14 1-Methyl-3,4-di-(2-Thienylcarbonyloxy)-2(1H)-Quinolinone(Formula 1.8 from Formula 1.02)

Obtained by acylation of 1-methyl 3,4-dihydroxy-2(1H)-quinolinone(Example 7, obtained from Formula 1.02, see Example 1) withthiophene-2-carboxylic acid using the procedure described in Example 11.That the expected product was obtained was confirmed by the spectraldata: MS(FAB): m/e 412(M.⁺ +1); NMR(CDCl₃): δ3.82 (s, 3H, CH₃ --N) ppm.

EXAMPLE 15 1-Methyl-3,4-di-(iso-Nicotinyloxy)-2(1H)-Quinolinone (Formula1.12 from Formula 1.02)

Obtained by acylation of 1-methyl-3,4-dihydroxy-2(1H)-quinolinone(Example 7, obtained from Formula 1.02, see Example 1) with isonicotinicacid using the procedure described in Example 11. That the expectedproduct was obtained was confirmed by the spectral data: MS(CI): m/e 402(M.⁺ +1); NMR(CDCl₃): δ3.87 (s, 3H, CH₃ --N), 8.80 (d, 2H, CH═N),8.90(d, 2H, CH═N) ppm.

EXAMPLE 16 1-Methyl-3,4-di-Benzoxy-2(1H)-Quinolinone (Formula 1.9 fromFormula 1.02)

Obtained by acylation of 1-methyl-3,4-dihydroxy-2(1H)-quinolinone(Example 7, obtained from Formula 1.02, see Example 1) with benzoylchloride using the procedure described in Example 11. That the expectedproduct was obtained was confirmed by the spectral data: MS(FAB): m/e400 (M.⁺ +1); NMR(DMSO): δ3.83 (s, 3H, CH₃ --N), 8.17 (m, 4H, ═CH--CO)ppm.

EXAMPLE 17 1-Methyl-3,4-di-Acetoxy-2(1H)-Quinolinone (Formula 1.13 fromFormula 1.02)

Obtained by acylation of 1-methyl-3,4-dihydroxy-2(1H)-quinolinone(Example 7, obtained from Formula 1.02, see Example 1) with aceticanhydride in pyridine and purifying the product by the proceduredescribed in Example 11. That the expected product was obtained wasconfirmed by the spectral data: MS(CI): m/e 276 (M.⁺ +1); NMR(DMSO):δ2.31 (s, 3H, CH₃ COO), 2.48(s, 3H, CH₃ COO), 3.70 (s, 3H, CH₃ --N) ppm.

EXAMPLE 18 1-Methyl-3,4-di-Propionoxy-2(1H)-Quinolinone (Formula 1.10from Formula 1.02)

Obtained by acylation of 1-methyl-3,4-dihydroxy-2(1H)-quinolinone(Example 7, obtained from Formula 1.02, see Example 1) with propionicanhydride in pyridine and purifying the product by the proceduredescribed in Example 11. That the expected product was obtained wasconfirmed by the spectral data: MS(FAB): m/e 304 (M.⁺ +1); NMR(CDCl₃):δ1.30 (t, 3H, CH₃), 1.36 (t, 3H, CH₃), 2.70 (m, 4H, CH₂ CO), 3.76(s, 3H,CH₃ --N) ppm.

EXAMPLE 19 1-Methyl-3,4-di-Methoxy-2(1H)-Quinolinone (Formula 1.11 fromFormula 1.02)

A suspension of 1-methyl-3,4-dihydroxy-2(1H)-quinolinone (0.2 gm,Example 7, obtained from Formula 1.02, see Example 1) in methylenechloride (5 ml) and methanol (5 ml) was treated with a solution ofexcess diazomethane in ether. The resulting solution was evaporated andthe residue was chromatographed on silica gel (5 gm). The major productof the reaction was eluted with 1% methanol/methylene chloride andcrystallized from ethyl acetate-hexane to give the title compound. Thatthe expected product was obtained was confirmed by the spectral data:MS: m/e 219 (M.⁺ +1); NMR(CDCl₃): δ3.71 (s, 3H, CH₃ --N), 3.94 (s, 3H,OCH₃), 4.22 (s, 3H, OCH₃) ppm.

EXAMPLE 20 3,4,7-Trihydroxy-8-methyl-1,2-benzopyrone (Formula 1.14 fromFormula 1.06)

Obtained from Formula 1.06 (Example 5) using the hydrolytic reductionprocedure described in Example 7.

EXAMPLE 21 3,4-Dihydroxy-1,2-benzothiopyrone (Formula 1.15 would beobtained from Formula 1.07)

The title compound would be obtained if the compound of Formula 1.07(Example 6) were to be reductively hydrolyzed using the proceduredescribed in Example 7.

BIOLOGICAL DATA Cell and Virus Culture

HeLa and Vero cell cultures were maintained in Eagles Minimal EssentialMedium which was supplemented with glutamine, penicillin, streptomycinand 10% fetal calf serum (10% EMEM). Stock cultures of HSV-2 (strain MSavailable from ATCC VR-540) were grown in and harvested from Vero cells.Viral stocks were titered in Vero cells according to establishedprocedures.

Plasmid Constructions

Plasmid pON 245^(ori-) contains the promoter of the HSV-1 thymidinekinase (tk) gene located immediately 5' of the E. coli lac Z gene. Inthis arrangement, the tk promoter controls transcription from thebacterial gene in transient expression assays. Additionally, an SV40polyadenylation signal is present at the 3' end of the lac Z gene toallow for the efficient translation of the mRNA in eucaryotic cells. Theexpression of beta galactosidase in a transient assay using pON245^(ori-) is dependent upon superinfection of the transfected cellswith HSV. Therefore, a compound which interferes with early steps of HSVreplication will also inhibit beta galactosidase production intransfected cells. For example, see U.S. application Ser. No. 07/435,491filed Sep. 5, 1989, the disclosure of which is incorporated herein byreference thereto.

Transient Expression of Beta Galactosidase in Transfected Cells

HeLa cells were seeded into 96 well microtiter plates and allowed togrow to 80% confluency (approximately 35000 cells/well). One halfmicrogram of plasmid pON 245^(ori-) DNA was introduced into the cells ofeach well by the DEAE Dextran precipitation technique (Grahman and Vander Eb, 1973). Four to six hours later, the cells were rinsed withHank's Balanced Salt Solution (HBSS), overlaid with 10% EMEM andincubated at 37° C. At 24 hrs post-transfection, cells were rinsed,overlaid with 10% EMEM again and re-incubated at 37° C. At 48 hrs.post-transfection, cells were rinsed and overlaid with either EMEMcontaining 2% fetal calf serum (2% EMEM), 2% EMEM containing HSV-2(strain MS, Multiplicity of Infection [moi]=5 pfu/cell) or 2% EMEMcontaining HSV-2 and the appropriate concentration of the compound to betested. Twenty-four hrs later, the cells were harvested and assayed forbeta galactosidase activity as described below.

Beta Galactosidase Assay

All determinations of beta galactosidase activity were performed in 96well microtiter plates. The intracellar level of beta galactosidaseactivity in each well was determined from cell lysates of the monolayercultures. Aliquots were assayed by incubation in the presence of betagalactosidase substrate, 4-methylumbelliferyl-β-D-galactoside (MUG, 125ug/ml, Sigma), for 2 hrs. The generation of fluorescent product wasquantified on a Microfluor microfluorimeter (Dynatech) after addition of0.1M glycine, pH 10.3 (Spaete and Mocarski, 1985). The inhibitoryactivity of a compound was plotted versus the concentration and an IC50value (the concentration of compound required to reduce betaglactosidase expression by 50%) was obtained for each compound tested.

Compound Toxicity Assay

Compounds which demonstrated a significant inhibitory activity in theHeLa cell beta galactosidase assay were assayed for their inhibitoryeffect on HeLa cell translation. HeLa cells were treated with inhibitorycompound for 24 hrs, after which levels of translational activity wereassayed.

For assay of translational activity, HeLa cultures were grown to 80%confluency in 96 well microtiter plates, treated with appropriateconcentrations of compound in 2% EMEM during an overnight incubation at37° C., then rinsed with HBSS and overlaid with 0.8 ml of 2% EMEMcontaining 8 uCi of tritiated leucine (³ H-LEU, 141 Cu/mMol, AmershamCorp., Arlington Heights Ill.). After a 1 hr incubation at 36.5° C., thecells were rinsed twice with phosphate buffered saline (PBS) and lysedin 400 ul/well of 1×PBS, 0.5% sodium dodecyl sulphate (SDS). After a 10min incubation at 36.5° C., the contents of the well were transferred toa well in a Millititer HA microfiltration plate (Millipore Corp.,Bedford, Mass.). The TCA insoluble proteins were precipitated onto thefilter disc by a 10 min fixation with 5% TCA, followed by filtrationunder vacuum and three 10 minute rinse with 95% ethanol. The filterswere dried at room temperature, cut from the milltitier plate andtransferred to scintillation vials. TCA precipitable counts were assayedin 5 ml of Scintisol (Isolab, Akron, Ohio). The inhibitory activity of acompound was plotted versus the concentration and an IC50 value (thatconcentration of the compound required to decrease cellulartranslational activity by 50%) was derived for each compound.

Analysis of In Vivo Efficacy

The in vivo assessment of anti-HSV efficacy was determined in theprophylactic guinea pig model of HSV infection described by Stansberryet al (1982). Dosing of guinea pigs was comprised of an initialtreatment with test compound given 24 hrs prior to virus infection andsubsequent administration of the compound every eight hours (T.I.D.) fora total of 10 days. Test compounds were administered subcutaneously in0.5% buffered methyl cellulose at a dose of 60 mg per kg body weight ofthe animal. Animals were monitored daily for the developement of genitallesions and neurological symptomology, both of which were recorded andcompared to the results obtained with parallel groups which receivedplacebo or acyclovir treatment. Efficacy was evaluated for each compoundby scoring the ability of the compound to ameliorate genital lesionsproduced by infection with HSV-2, strain MS, expressed as Maximum Lesionscores (MLS) on a scale of 1 (least lesions) to 4 (severe lesions).

In Vitro Anti-HSV Activity

The in-vitro anti-HSV activity of compounds of this invention is setforth in Table I. More than one number for a particular entry indicatesthe results for additional tests.

                  TABLE I                                                         ______________________________________                                                  ANTI-HSV ACTIVITY                                                                             CYTOTOXICITY                                                  HSV-β-GAL ASSAY                                                                          .sup.3 H-LEU ASSAY                                  FORMULA   IC.sub.50 (μg/ml)                                                                          IC.sub.50 (μg/ml)                                ______________________________________                                        1.02      7               27                                                  1.03      7               40                                                  1.04      <3, 2.5         37                                                  ______________________________________                                    

In-Vivo Anti-HSV Activity

The compound of Formula 1.04 was inactive when tested in-vivo using thein-vivo protocol set forth above.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention and all suchmodifications are intended to be included within the scope of theclaims.

What is claimed is:
 1. The compound of the formula ##STR29##
 2. Thecompound of the formula ##STR30##
 3. A pharmaceutical compositioncomprising an effective amount of a pharmaceutically acceptable carrierand an effective amount of a compound of claim
 1. 4. A pharmaceuticalcomposition comprising an effective amount of a pharmaceuticallyacceptable carrier and an effective amount of a compound of claim
 2. 5.A method of treating a viral infection in a patient in need of suchtreatment comprising administering an effective amount of a compound ofclaim 1 to said patient.
 6. A method of treating a viral infection in apatient in need of such treatment comprising administering an effectiveamount of a compound of claim 2 to said patient.